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Etude par microscopie électronique des conformations structurales adoptées par la Formine durant l'assemblage de filaments d'actine

Abstract : Formins are key regulators of the assembly of actin filaments in cells. Formins induce the nucleation and rapid elongation of actin filaments. The activity of formins thus participates in the generation of functional structures such as filopodia, stress fibers or the cytokinetic ring. Hence, formins are involved in fundamental cellular processes such as migration or cell division. At the molecular level, formins are homodimers capable of interacting with the barbed ends of elongating actin filaments via their FH2 domains. The conformational changes involved in tracking the barbed ends have never been directly observed. However, a set of biochemical, biophysical and structural biology studies evidence that the FH2 dimer moves stepwise at the barbed end, following an equilibrium between two states : "closed" and "opened". In the closed state, no new actin subunit can be added to the barbed end, while it is allowed in the opened state. Several models, under discussion, describe the structural nature of these two states and how they alternate. One can then evoke the « stair-stepping » model and the « stepping second » model.My thesis aims at elucidating the debate about the conformational states adopted by formin at the barbed end, focusing on the mammalian formin mDia1. Our goal was undertaken using electron microscopy. So far, formins bound to the barbed ends of actin has not been described using electron microscopy. Indeed, to pursue single particle analysis by EM, a sufficient density of objects of interest randomly oriented have to be displayed within an image. Formins bound to actin rapidly generate long filaments, preventing the obtention of enough short filaments exhibiting formins at their barbed ends. Thus, a bottleneck had to be overcome to make the samples suitable for single particle structural analysis. A sonication approach was chosen to produce short actin filaments. A first 3D reconstruction of the FH2 dimer-actin complex was obtained from negative stain electron microscopy samples. This envelope corresponds to the open state of the « stair stepping » model. After optimizing the image analysis protocol, a second 3D conformation of the complex could be determined, corresponding to the closed state of the so-called « stair stepping » model, which was minoritary. A further 2D study also revealed conformational variations within the open state. These observations led to the hypothesis that, for extreme conformations, the FH2 dimer can be found not only at the barbed end but also on the body of the actin filaments. The search for such a configuration could be validated by 2D analyses and a preliminary 3D density of an FH2 dimer complex encircling the body of an actin filament. We have adapted our samples to cryo-electron microscopy. Graphene-Oxide was added to the surface of the EM grids to preserve a sufficient actin filament ends density. A limited quantity of filament ends could be analyzed through 2D and 3D classifications. It thus confirms the non-artefactual nature of the previously observed open state.In addition, through a collaborative project, I also showed the synergistic action of Formin with Arp2/3 in the presence of Spin90 by highlighting the formation of a tetramer.
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Submitted on : Tuesday, September 14, 2021 - 3:10:13 PM
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Julien Maufront. Etude par microscopie électronique des conformations structurales adoptées par la Formine durant l'assemblage de filaments d'actine. Biologie cellulaire. Université Paris sciences et lettres, 2021. Français. ⟨NNT : 2021UPSLS049⟩. ⟨tel-03344046⟩

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